Mycobacterium tuberculosis (Mtb) infects approximately 1.7 billion people worldwide, with over 10 million people developing active tuberculosis (TB) disease each year. The vast majority of infected individuals do not develop symptomatic disease and are considered to have latent Mtb infection (LTBI). The single greatest risk factor for development of active TB disease is co-infection with human immunodeficiency virus (HIV). Untreated HIV infection is associated with systemic immune activation, which is considered to be a significant driver of high levels of T cell activation, apoptosis of bystander T cells, and overall loss of CD4 T cells that is characteristic of chronic HIV infection. CD4 T cells are critical for successful immune control of Mtb infection, yet the precise mechanisms whereby HIV infection perturbs CD4 T cell immunity to Mtb and accelerates progression to TB disease have not been well defined. The focus of this proposal is to define potential mechanisms of HIV-associated dysregulation of Mtb-specific CD4 T cell responses in individuals with HIV/Mtb co-infection. We and others have shown that, compared with HIV-uninfected individuals, Mtb-specific CD4 T cells from HIV-infected individuals have diminished cytokine production, a heightened level of activation, profoundly impaired proliferative capacity, and an increased susceptibility to cell death following Mtb antigen stimulation, thus providing compelling evidence of Mtb-specific CD4 T cell dysfunction in the setting of HIV infection. We propose to test the hypotheses that (1) HIV-associated immune activation contributes to functional impairment of Mtb-specific CD4 T cell responses, and (2) HIV infection dysregulates immune signaling networks associated with cell death and T cell dysfunction in Mtb-specific CD4 T cells. Using samples collected from a well-characterized longitudinal cohort of HIV-infected individuals with LTBI in Kenya, sampled prior to and multiple times over the first year of antiretroviral therapy (ART), we will perform a detailed evaluation of the relationship between HIV viral load, immune activation, and the phenotypic and functional signatures of Mtb-specific CD4 T cells in individuals with HIV/Mtb co-infection. We will then utilize state-of-the- art technology to conduct single-cell RNA-sequencing of Mtb-specific CD4 T cells from HIV-infected and HIV- uninfected individuals with LTBI, to define novel immune signaling networks that are dysregulated in Ag- specific CD4 T cells from HIV-infected individuals. Identification of signaling pathways that are differentially expressed in Ag-specific CD4 T cells from HIV-infected individuals will open up new avenues of research in host-directed therapies to promote the generation and maintenance of protective Mtb-specific T cell responses that can mediate sustained control of Mtb infection and prevent progression to active TB disease.